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WO2025004049A1 - Traitement d'infections provoquées par des pathogènes intracellulaires - Google Patents

Traitement d'infections provoquées par des pathogènes intracellulaires Download PDF

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Publication number
WO2025004049A1
WO2025004049A1 PCT/IL2024/050636 IL2024050636W WO2025004049A1 WO 2025004049 A1 WO2025004049 A1 WO 2025004049A1 IL 2024050636 W IL2024050636 W IL 2024050636W WO 2025004049 A1 WO2025004049 A1 WO 2025004049A1
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alkyl
hydrogen
composition
cycloalkyl
alkoxy
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Ohad GAL-MOR
Boaz ADANI
David Margulis
Leila MOTIEI
Barr HAIM
Shurrush KHRIESTO
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Sheba Impact Ltd
Yeda Research and Development Co Ltd
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Sheba Impact Ltd
Yeda Research and Development Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/138—Aryloxyalkylamines, e.g. propranolol, tamoxifen, phenoxybenzamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/351—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom not condensed with another ring
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4704—2-Quinolinones, e.g. carbostyril
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A61P31/06—Antibacterial agents for tuberculosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals

Definitions

  • the present invention in some embodiments thereof, relates to treatment of infections caused by intracellular pathogens.
  • This compartment offers protection from the host's humoral immunity, sequestration from neutrophils, and access to nutrients that may be scarce extracellularly 4 ’ 5 .
  • intracellular bacterial pathogens have evolved to sophisticatedly manipulate host cells to access their preferred niches within targeted cells.
  • bacteria are contained within a plasma membrane-derived vacuole, such as phagosomes or endosomes.
  • Vacuolar intracellular bacteria like Salmonella enterica or Mycobacterium tuberculosis remain within modified vacuoles, while intracellular cytosolic bacteria, like Listeria monocytogenes or Shigella spp., rupture the vacuole and reside in the host cytosol 4-7 .
  • Intracellular pathogens have co-evolved with hosts and developed a striking ability to manipulate and subvert multiple host functions and pathways to facilitate their survival and transmission.
  • the process by which intracellular bacteria hijack host cells can be divided into four distinct stages: adhesion, internalization, survival/prolif eration, and dissemination.
  • adhesion adhesion
  • internalization internalization
  • survival/prolif eration survival/prolif eration
  • dissemination Although different pathogens use distinct strategies to subvert host pathways, eliminating the replicative niche of pathogens by regulated cell death of host cells serves as a universal defense mechanism against a wide array of intracellular pathogens.
  • PAMPs Pathogen- associated molecular patterns
  • PRRs pattern-recognition receptors
  • This lytic form of cell death removes the replicative niche for intracellular pathogens by inducing the formation of large pores in the plasma membrane. This process releases highly inflammatory cytoplasmic contents, including proinflammatory cytokines and damage-associated molecular patterns (DAMPs), which recruit and activate immune cells to combat the infection 10 .
  • DAMPs damage-associated molecular patterns
  • a method of treating or preventing an infection by an intracellular pathogen in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising at least one compound represented by Formula I, II, III, IV, V, VI or VII, as described herein in any of the respective embodiments and any combination thereof.
  • a method of inhibiting growth of an intracellular pathogen or invasion thereof into host cells comprising contacting the pathogen with a therapeutically effective amount of a pharmaceutical composition comprising at least one compound represented by Formula I, II, III, IV, V, VI or VII as described herein in any of the respective embodiments and any combination thereof.
  • a method of treating or preventing an infection by an intracellular pathogen in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a compound which induces programmed cell death (PCD) in infected host cells of the intracellular pathogen, thereby treating or preventing the infection.
  • PCD programmed cell death
  • a method of inhibiting growth of an intracellular pathogen or invasion thereof into host cells comprising contacting the pathogen with a therapeutically effective amount of a pharmaceutical composition comprising a compound which induces PCD in host cells infected with an intracellular pathogen, thereby inhibiting growth of the intracellular pathogen or invasion thereof into host cells.
  • the method is performed in vitro.
  • the method is performed in vivo.
  • a method of identifying an agent against intracellular pathogens comprising:
  • the cis-acting regulatory element is a promoter.
  • the promoter comprises the ssek3 promoter.
  • the method further comprises determining viability of the host cells in the presence of the agent in comparison to an absence thereof, and wherein substantially the same viability is indicative that the agent is safe.
  • the host cells are epithelial cells.
  • a pharmaceutical composition comprising at least one compound represented by Formula I, II, III, IV, V, VI or VII as described herein in any of the respective embodiments and any combination thereof, for use in treating or preventing an infection by an intracellular pathogen in a subject in need thereof.
  • a pharmaceutical composition comprising a compound which induces PCD in host cells of an intracellular pathogen for use in treating or preventing an infection by the intracellular pathogen in a subject in need thereof.
  • the intracellular pathogen is mycobacterium tuberculosis
  • the compound is represented by Formula I,
  • the compound is not a Src inhibitor.
  • the compound is not bactericidal.
  • the intracellular pathogen is selected from the group consisting of bacterial pathogen, viral pathogen, fungal pathogen and a protozoan pathogen.
  • the intracellular pathogen comprises bacteria.
  • the bacteria is Gram positive.
  • the bacteria is Gram negative.
  • the bacteria is of a genus selected from the group consisting of Listeria monocytogenes, Salmonella enterica serovars (e.g., Salmonella Typhi), Mycobacterium tuberculosis, Chlamydia trachomatis, Rickettsia species and Legionella pneumophila.
  • the bacteria comprises a vacuolar intracellular bacterium.
  • the bacteria is Salmonella enterica or Mycobacterium tuberculosis.
  • the bacteria comprises an intracellular cytosolic bacterium.
  • the bacteria is Listeria monocytogenes or Shigella spp.
  • the infection causes tuberculosis (TB), chlamydia, listeriosis, invasive salmonellosis, legionnaire’s disease, Rocky Mountain spotted fever (R. rickettsii) or typhus (R. prowazekii).
  • TB tuberculosis
  • chlamydia listeriosis
  • invasive salmonellosis legionnaire’s disease
  • Rocky Mountain spotted fever R. rickettsii
  • typhus R. prowazekii
  • n is 0 or 1 ;
  • R 1 and R 2 are each independently selected from hydrogen and alkyl, preferably a lower alkyl of 1 to 4 carbon atoms in length, wherein at least one of R 1 and R 2 is the alkyl;
  • R 3 -R 14 are each independently selected from hydrogen, alkyl, cycloalkyl, halo, haloalkyl, hydroxy, alkoxy, thiol, thioalkoxy, amine, cyano and nitro;
  • R 15 -R 24 are each independently selected from hydrogen, alkyl, cycloalkyl, amine, halo, hydroxy, thiol, alkoxy, and thioalkoxy; and
  • R25 is selected from hydrogen, alkyl and cycloalkyl.
  • n is i.
  • R 1 and R 2 are each independently selected from methyl, ethyl, propyl and isopropyl.
  • R 1 and R 2 are each methyl; or Ri is methyl and R 2 is ethyl; or Ri is isopropyl and R 2 is hydrogen.
  • R 3 -R 13 are each hydrogen.
  • R 14 is alkyl, preferably a lower alkyl (e.g., methyl).
  • each of R 15 - R 19 is hydrogen.
  • At least one of R 15 -R 19 preferably at least one of R 15 , R 17 or R 19 is selected from alkyl (preferably lower alkyl), fluoro, and alkoxy (preferably lower alkoxy).
  • R 15 is alkoxy (preferably lower alkoxy).
  • At least one of R 20 -R 24 preferably at least one of R 20 , R 22 or R 24 is selected from amine (preferably a tertiary amine), fluoro, and alkoxy (preferably lower alkoxy).
  • R 22 is selected from amine (preferably a tertiary amine), fluoro and alkoxy (preferably lower alkoxy).
  • the compound is:
  • PCM-1013431 also referred to herein as C4
  • the compound is selected from compounds denoted as 21, 30, 46 and 50 (see, hereinbelow and FIG. 10B).
  • the compound is selected from compounds denoted as 12, 15, 21, 24, 27, 28, 29, 30, 31, 46, 47, 48, 50, 51 and 53 (see, hereinbelow and FIG. 10B).
  • the compound is represented by Formula II: wherein:
  • R 30 and R 31 are each independently selected from hydrogen, alkyl and cycloalkyl
  • R 32 -R 36 are each independently selected from hydrogen, alkyl, cycloalkyl, halo, haloalkyl, hydroxy, alkoxy, thiol, thioalkoxy, amine, cyano and nitro, provided that at least one of R 32 -R 36 , preferably at least one of R 32 , R 34 and R 36 is halo; and
  • A is selected from: wherein the curved line represents an attachment point to NR30-; and R 37 and R38 are each independently selected from hydrogen alkyl and cycloalkyl,
  • R 30 and R 31 are each hydrogen.
  • A is least two of R 32 -R 36 , preferably at least two of R 32 , R 34 and R 36 , are each halo, preferably chloro. According to some embodiments of any of the embodiments described herein, A is , two of R 32 , R 34 and R 36 , are each halo, preferably chloro and the third is hydrogen, and R33 and R35 are each hydrogen.
  • A is and no more than one of R 32 -R 36 , preferably one of R 32 , R 34 and R 36 , is halo, preferably chloro.
  • A is , R 32 is halo, preferably chloro, and R33-R 36 are each hydrogen.
  • R 37 and R38 are each independently selected from hydrogen and alkyl.
  • the compound is selected from:
  • the compound is represented by Formula III:
  • R 40 -R 44 are each independently selected from hydrogen, alkyl, cycloalkyl, halo, hydroxy, alkoxy, thiol, thioalkoxy, amine, provided that at least one of R 40 -R 44 , preferably R 40 , is a cycloalkyl; and R 45 -R 52 are each independently selected from hydrogen, alkyl, cycloalkyl, halo, hydroxy, alkoxy, thiol, thioalkoxy, amine, provided that at least one of R 45 -R 50 is selected from hydroxy, thiol, alkoxy and thioalkoxy,
  • R 40 is a cycloalkyl and is preferably cyclopentyl.
  • R 41 -R 44 are each hydrogen.
  • R 47 is hydroxy
  • R 45 , R 46 and R 48 -R 50 are each hydrogen.
  • R 51 is hydrogen
  • R 52 is hydrogen or an alkyl, preferably a bulky lower alkyl such as t-butyl.
  • the compound is: , and is also referred to herein as C2.
  • the compound is represented by Formula IV :
  • R 61 -R 64 are each independently selected from hydrogen, alkyl, cycloalkyl, halo, haloalkyl, amine, hydroxy, alkoxy, thiol, thioalkoxy;
  • R 65 and R66 are each independently selected from hydrogen, alkyl and cycloalkyl
  • R 67 -R 78 are each independently selected from hydrogen, alkyl, cycloalkyl, halo, haloalkyl, amine, hydroxy, alkoxy, thiol and thioalkoxy;
  • R 80 represents one or more (1, 2, 3, 4 or 5) substituents on the phenyl ring, or is absent, wherein each of these substituents, if present, is independently selected from alkyl, cycloalkyl, halo, haloalkyl, amine, hydroxy, alkoxy, thiol, and thioalkoxy,
  • At least one of R 61 -R 64 is selected from alkyl and halo, and is preferably a trihaloalkyl.
  • each of R 65 and Ree is hydrogen.
  • R 67 -R 70 are each hydrogen.
  • At least one of R 71 -R 76 is hydroxy.
  • R 71 -R 73 , R 75 and R 76 are each hydrogen and R 74 is hydroxy.
  • R77 and R78 are each hydrogen and/or R 79 is absent.
  • the compound is:
  • the compound is represented by Formula V : wherein:
  • R 80 and R 81 are each independently selected from hydrogen, alkyl and cycloalkyl
  • R 82 -R 86 are each independently selected from hydrogen, alkyl, cycloalkyl, halo, haloalkyl, amine, hydroxy, alkoxy, thiol, and thioalkoxy;
  • R 87 -R 90 are each independently selected from hydrogen, alkyl, cycloalkyl, halo, haloalkyl, amine, hydroxy, alkoxy, thiol, and thioalkoxy;
  • R 91 -R 94 are each independently selected from hydrogen, alkyl, cycloalkyl, halo, haloalkyl, amine, hydroxy, alkoxy, thiol, and thioalkoxy;
  • R 95 -R 98 are each independently selected from hydrogen, alkyl, cycloalkyl, halo, haloalkyl, amine, hydroxy, alkoxy, thiol, and thioalkoxy,
  • R 80 and R 81 are each hydrogen.
  • R 82 and R 83 are each hydrogen. According to some embodiments of any of the embodiments described herein, at least one of R 84 -R 86 , preferably R 84 , is hydroxy.
  • At least one of R 87 -R 90 is hydroxy.
  • each of R91- R94 is hydrogen.
  • At least one, or at least two of R 95 -R 98 is an alkyl, preferably a lower alkyl.
  • R 96 and R 97 are each independently an alkyl, preferably a lower alkyl.
  • the compound is:
  • the compound is represented by Formula VI:
  • R 100 is hydrogen, alkyl or cycloalkyl
  • R 101 -R 105 are each independently selected from hydrogen, alkyl, cycloalkyl, aryl, halo, haloalkyl, amine, hydroxy, alkoxy, thiol, thioalkoxy, heteroaryl, cyano, and nitro, wherein at least one of R 101 -R 105 , preferably R 101 , is aryl;
  • R 106 -R 109 are each independently selected from hydrogen, alkyl, cycloalkyl, halo, haloalkyl, amine, hydroxy, alkoxy, thiol and thioalkoxy;
  • R 110 -R 113 are each independently selected from hydrogen, alkyl, cycloalkyl, halo, haloalkyl, amine, hydroxy, alkoxy, thiol and thioalkoxy, or, alternatively, two of R 110 -R 113 form together a substituted or unsubstituted cyclic ring (alicyclic, heteroalicyclic, aryl or heteroaryl); and
  • R 114 -R 117 are each independently selected from hydrogen, alkyl, cycloalkyl, halo, haloalkyl, amine, hydroxy, alkoxy, thiol and thioalkoxy,
  • R100 is hydrogen
  • At least one of R 101 -R 105 is aryl, preferably phenyl.
  • R 101 is the aryl, and wherein optionally R 102 -R 105 are each hydrogen.
  • At least two of R 110 -R 113 form together a substituted or unsubstituted cyclic ring, preferably a substituted or unsubstituted heteroaryl.
  • Rm and Rm form together a substituted or unsubstituted imidazole.
  • R106-R109 are each hydrogen and/or R 114 -R 117 are each hydrogen.
  • the compound is: , also referred to herein as C6.
  • the compound is represented by Formula VII:
  • X is O, S or NR 120 ;
  • R 119 and R 120 are each independently selected from hydrogen, alkyl or cycloalkyl;
  • R 121 -R 125 are each independently selected from hydrogen, alkyl, cycloalkyl, aryl, halo, haloalkyl, amine, hydroxy, alkoxy, aryloxy, thioaryloxy, thiol, thioalkoxy, heteroaryl, cyano and nitro, or, alternatively or in addition, two of R 121 -R 125 form together a substituted or unsubstituted cyclic ring (alicyclic, heteroalicyclic, aryl or heteroaryl); and
  • R 126 -R 129 are each independently selected from hydrogen, alkyl, cycloalkyl, halo, haloalkyl, amine, hydroxy, alkoxy, thiol, thioalkoxy, heteroalicyclic, heteroaryl, and aryloxy, provided that at least one of R 121 -R 125 and at least one of R 126 -R 129 is other than hydrogen.
  • X is NR 120 .
  • R 119 and R 120 are each hydrogen.
  • At least one of R 121 -R 125 is alkoxy or aryloxy.
  • At least one of R 126 -R 129 is alkoxy, aryloxy or a heteroaryl.
  • At least two of R 121 -R 125 form together a cyclic ring, preferably a heteroalicyclic ring.
  • R 121 and R 122 form together the cyclic ring.
  • At least two of R 126 -R 129 are each independently an alkoxy. According to some embodiments of any of the embodiments described herein, at least two of R 126 -R 129 are each independently an alkoxy.
  • the compound is represented by Formula Vila: wherein:
  • R 121 and R 122 form together the cyclic ring, preferably the heteroalicyclic ring
  • R 130 and R 131 are each independently selected from alkyl, cycloalkyl, and heteroalicyclic.
  • R 121 and R 122 form together a dioxolane.
  • At least one of R 123 -R 125 , preferably R 125 is halo, preferably chloro.
  • R 130 and R 131 each independently comprise a heteroalicyclic.
  • R 130 is a heteroalicyclic, preferably tetrahydropyrane.
  • R 131 is an alkyl substituted by a heteroalicylic, preferably a piperazine.
  • the compound is:
  • At least one of R 121 -R 125 is aryloxy, preferably heteroaryloxy.
  • R 123 is the aryloxy
  • At least one of R 126 -R 129 is a heteroaryl, preferably furyl, more preferably a substituted furyl.
  • R 127 is the heteroaryl
  • the compound is represented by Formula Vllb: wherein: at least one of R 121 -R 125 is the aryloxy, preferably a heteroaryloxy; and
  • R 132 -R 134 are each independently selected from hydrogen, alkyl, cycloalkyl, halo, haloalkyl, amine, heteroalicyclic, heteroaryl, cyano and nitro.
  • R 123 is heteroaryloxy, preferably 7-triazolopyridinoxy.
  • at least one of R 121 , R 122 , R 124 and R 125 , preferably R 124 is alkyl, preferably methyl.
  • At least one of R 13 2-R 13 4, preferably R 13 2, is an alkyl, preferably a substituted alkyl.
  • the alkyl is substituted by a moiety that comprises an amine and/or SO 2 , preferably a hydrocarbon interrupted and/or substituted by an amine and/or SO 2 .
  • the alkyl is -CH 2 -NH-CH 2 -CH 2 -SO 2 -CH 3 .
  • the compound is: also referred to herein as C5.
  • R 123 is other than hetero aryloxy; and/or R 132 is other than an alkyl substituted by a moiety that comprises an amine and/or SO 2 , or a hydrocarbon interrupted and/or substituted by an amine and/or SO 2 .
  • FIG. 1 is an illustration showing an embodiment of high-throughput screening (HTS) for compounds having activity against intracellular pathogens.
  • HTS high-throughput screening
  • FIGs. 2A-B present a scheme showing an embodiment of the pipeline and selection stages of the HTS illustrated in FIG. 1 (FIG. 2A) and the chemical structures of exemplary top hits identified in the HTS (FIG. 2B);
  • FIG. 3 shows an inhibitory effect of compounds identified in the HTS on Salmonella infection
  • FIG. 4 shows the cytotoxic activity (against HeLa and HB2 cells) of compounds identified in the HTS
  • FIG. 5 shows the inhibitory activity of compounds identified in the HTS in a dose-response assay
  • FIG. 6 shows that compounds identified in the HTS inhibit mainly Salmonella intracellular replication rather than invasion
  • FIG. 7 shows activity of compounds identified in the HTS in inhibiting intracellular growth of S. Typhimurium in a mouse bone-marrow derived macrophages (BMDMs);
  • FIG. 8 shows that compounds identified in the HTS inhibit the intracellular growth of Listeria monocytogenes'
  • FIG. 9 shows that compounds identified in the HTS trigger programmed cell death in infected host cells
  • FIGs. 10A-B present data obtained in Structure Activity Relationship (SAR) studies of compounds PCM-0103431 and PCM-0001349 (FIG. 10A) and the chemical structures of exemplary structural analogs used in the SAR studies (FIG. 10B).
  • SAR Structure Activity Relationship
  • the present invention in some embodiments thereof, relates to treatment of infections caused by intracellular pathogens.
  • Intracellular bacterial pathogens including Mycobacterium, Listeria, and Salmonella continue to threaten human and animal health globally, affecting millions of lives and causing substantial economic loss.
  • the ability of these clinically important pathogens to invade and localize within host cells provide them protection from both antibacterials and the host immune system, which can lead to chronic untreatable infections.
  • the constant emergence and spreading of new multi- and extreme-drug resistant strains significantly limit and often completely prevent antibiotic therapy.
  • novel approaches and new treatment options are urgently required to fight intracellular resistant pathogens.
  • HTS high-throughput screening
  • pathogens like Salmonella in epithelial and phagocytic human host cells at ⁇ M concentrations.
  • SAR Structure activity relationship
  • HDTs In contrast to conventional antibiotics that directly act on bacteria, it is suggested that the identified compounds act as host-directed therapies HDTs that enhance a potentially broad response against bacteria, and render host cells less-permissive for bacterial growth.
  • HDTs have several important advantages over conventional antibiotic therapy, including: (i) HDTs can be effective against MDR infections; (ii) unlike antibiotics that directly target bacteria, HDTs act against intracellular pathogens by either potentiating host defenses or sensitizing internalized bacteria to immune clearance; (iii) in absence of direct selective pressure on the pathogen, HDT are less likely to develop drug resistance; (iv) HDTs have the potential to target metabolically inactive, non-replicating bacteria, which are tolerant to conventional therapies; and (v) potential synergies with HDTs and antibiotics acting on different pathways. Indeed, recent advances in HDTs have provided promising approaches to reduce or eliminate intracellular bacterial infections, by targeting the host factors, that can restrict the replication and persistence of pathogens inside the cell.
  • a method of treating or preventing an infection by an intracellular pathogen in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising at least one compound represented by Formula I, II, III, IV, V, VI or VII, as described herein in any of the respective embodiments and any combination thereof.
  • a method of inhibiting growth of an intracellular pathogen or invasion thereof into host cells comprising contacting the pathogen with a therapeutically effective amount of a pharmaceutical composition comprising at least one compound represented by Formula I, II, III, IV, V, VI or VII, as described herein in any of the respective embodiments and any combination thereof.
  • inhibiting the growth of an intracellular pathogen refers to inhibiting survival and/ or replication of an intracellular pathogen during host cell infection.
  • the phrase “inhibiting invasion to host cells” refers to inhibiting the entry or penetration of the pathogen into the host cell. This can be determined by a bioluminescence assay, microscopy, CFU counting and by other methods.
  • the inventors have developed a bioluminescence-based method, in which the pathogen expresses a reporter luciferase system under the control of a regulatory element that is induced only when the pathogen is in the host cell (but not in the extracellular environment), the intensity of the bioluminescence signal is proportionate to the amount of the intracellular pathogen. That is, the more pathogen invades, survives or replicate intracellularly, the higher the intensity of the bioluminescence signal.
  • inhibiting refers to a decrease of at least 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 95 % or complete inhibition (i.e., 100 %) in the survival, growth, or replication of the intracellular pathogen or invasion thereof as compared to that of an identical pathogen in an identical host system under the same conditions but without the compound or agent.
  • a method of treating or preventing an infection by an intracellular pathogen in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a compound which induces programed cell death in host cells infected by an intracellular pathogen, thereby treating or preventing the infection.
  • PCD programed cell death
  • caspase dependent cell death such as apoptosis and pyroptosis
  • non-caspase dependent cell death such as necroptosis, ferrotosis, and autophagy
  • induction refers to an increase of at least 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 95 % or more (e.g., at least 2, 3, 5, 10 fold) in a PCD signal in response to the compound or agent as compared to that in the absence thereof.
  • a method of inhibiting growth of an intracellular pathogen or invasion thereof into host cells comprising contacting the pathogen with a therapeutically effective amount of a pharmaceutical composition comprising a compound which induces PCD in host cells of the intracellular pathogen, thereby inhibiting growth of the intracellular pathogen or invasion thereof into host cells.
  • composition comprising at least one compound represented by Formula I, II, III, IV, V, VI or VII , as described herein in any of the respective embodiments and any combination thereof for use in treating or preventing an infection by an intracellular pathogen in a subject in need thereof.
  • composition comprising a compound which induces PCD in host cells of an intracellular pathogen for use in treating or preventing an infection by the intracellular pathogen in a subject in need thereof.
  • treating refers to curing, reversing, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of a pathogen infection.
  • preventing refers to preventing pathogen infection or the deleterious effects (e.g., symptoms) of a pathogen infection.
  • administer refers to injecting, implanting, absorbing, ingesting, or inhaling a compound or agent described herein, or a pharmaceutical composition thereof.
  • Subjects which may be treated according to this aspect of the present invention include animal subjects - e.g. humans; or any mammalian host; or plants which may suffer from a disease associate with infection by intracellular pathogens.
  • the infection is an acute infection.
  • the infection is a chronic infection.
  • intracellular pathogen refers to a pathogen that may belong to a diverse range of life forms including bacteria, viruses, fungi, protozoa, and parasitic worms that cause disease to a host infected therewith. Facultative or obligatory intracellular pathogens are characterized by their ability to invade and reside within host cells, often evading the host immune system and causing various diseases.
  • Intracellular Bacteria can be Gram positive or Gram negative
  • Listeria monocytogenes' Causes listeriosis. Invades and replicates within macrophages and other host cells, spreading cell-to-cell
  • Salmonella enterica e.g., Salmonella enterica serovar Typhi
  • Salmonella enterica serovar Typhi Causes typhoid fever and salmonellosis.
  • Mycobacterium tuberculosis' causess tuberculosis.
  • Chlamydia trachomatis Causes chlamydia.
  • Legionella pneumophila Causes Legionnaires' disease. Survives and replicates within amoebae in the environment and macrophages in humans.
  • the bacteria is of a genus selected from the group consisting of Listeria monocytogenes, Salmonella enterica (e.g., Salmonella enterica serovar Typhi), Mycobacterium tuberculosis, Chlamydia trachomatis, Rickettsia species and Legionella pneumophila.
  • Salmonella enterica e.g., Salmonella enterica serovar Typhi
  • Mycobacterium tuberculosis e.g., Salmonella enterica serovar Typhi
  • Chlamydia trachomatis e.g., Rickettsia species and Legionella pneumophila.
  • the bacteria reside within an intracellular vacuoles containing bacteria.
  • the bacteria is Salmonella enterica or Mycobacterium tuberculosis.
  • the bacteria reside intracellularly in its host cells cytosol.
  • the bacteria is Listeria monocytogenes or Shigella spp.
  • the infection causes tuberculosis (TB), chlamydia, listeriosis, invasive salmonellosis, legionnaire’s disease, Rocky Mountain spotted fever (R. rickettsii) or typhus (A. prowazekii).
  • TB tuberculosis
  • chlamydia listeriosis
  • invasive salmonellosis legionnaire’s disease
  • Rocky Mountain spotted fever R. rickettsii
  • typhus A. prowazekii
  • Intracellular Viruses It will be appreciated that all viruses are intracellular pathogens as they require host cell machinery for replication. Following are some examples: Human Immunodeficiency Virus (HIV): Causes AIDS. Infects CD4+ T cells, macrophages, and dendritic cells; Influenza virus: Causes the flu. Infects respiratory epithelial cells; Hepatitis B virus (HBV): Causes hepatitis B. Infects liver cells (hepatocytes); Herpes Simplex Virus (HSV): Causes oral and genital herpes.
  • HCV Human Immunodeficiency Virus
  • VZV Varicella-Zoster Virus
  • Coronavirus e.g., SARS-CoV2 causes various forms of C0VID19. Infects respiratory epithelial cells, endothelial cells, gastrointestinal cells and more.
  • Intracellular Fungi Histoplasma capsulatunv. Causes histoplasmosis. Survives within macrophages. Cryptococcus neoformans'. Causes cryptococcosis, primarily in immunocompromised individuals. Can survive intracellularly in macrophages;
  • Intracellular Protozoa Plasmodium species (e.g., Plasmodium falciparum)'.
  • Parasitic Worms Intracellular Helminths. While most helminths are extracellular, some have intracellular stages during their lifecycle; Trichinella spiralis'.
  • the virus is selected from the group consisting of Adeno-associated virus (AAV), Polyomavirus, SV40, Papillomavirus, Adeonovirus, Herpes simplex virus, EBV, VZV, Poliovirus, Rhinovirus, Hepatitis A, B, and C virus, Yellow fever virus, Influenza virus, Measles virus, Reovirus, Rotavirus virus, HIV, and SARS virus.
  • AAV Adeno-associated virus
  • Polyomavirus Polyomavirus
  • SV40 SV40
  • Papillomavirus Adeonovirus
  • Herpes simplex virus Herpes simplex virus
  • EBV VZV
  • Poliovirus Poliovirus
  • Rhinovirus Rhinovirus
  • Hepatitis A, B, and C virus Yellow fever virus
  • Influenza virus Measles virus
  • Reovirus Rotavirus virus
  • HIV HIV
  • SARS virus SARS virus
  • the protozoa is selected from the group consisting of Trypanosoma cruzi, Leishmania, Plasmodium, Polypodium, Cryptosporidium parvum, and Toxoplasma gondii.
  • the fungus is selected from the group consisting of Histoplasma capsulatum, Cryptococcus neoformans, Blastomyces dermatitidis , and Pneumocystis jirovecii.
  • the virus is selected from the group consisting of Salmonella, Chlamydia, Rickettsia, Coxiella, Mycobacterium, and listeria.
  • the intracellular pathogen is Mycobacterium tuberculosis, and the compound is represented by Formula I, II, III, IV, V or VI, or by VII, provided that the compound is not:
  • the intracellular pathogen is Mycobacterium tuberculosis, and the compound is represented by Formula I, II, III, IV, V or VI, or by Formula Vila.
  • the compound is represented by Formula I, II, III, IV, V or VI, or by Formula Vila.
  • the compound is not a Src inhibitor.
  • Src inhibitor refers to a class of compounds (some are FDA approved drugs, e.g., Dasatinib, Bosutinib, Saracatinib (AZD0530) and KX2-391) that target and inhibit the activity of Src family kinases (SFKs).
  • Src kinases are a group of non-receptor tyrosine kinases that play crucial roles in various cellular processes, including proliferation, differentiation, survival, and migration. Dysregulation of Src kinases has been implicated in the development and progression of several cancers and other diseases. Src inhibitors function by binding to the ATP- binding site of Src kinases, preventing their activation and subsequent phosphorylation of downstream targets.
  • the compound is not bactericidal.
  • bacteriaicidal refers to refers to compounds or agents that kill bacteria, effectively eliminating bacterial populations rather than merely inhibiting their growth.
  • a method of identifying an agent against intracellular pathogens comprising:
  • agent refers to a chemical compound which can be naturally occurring or synthetic.
  • the agent thus identified can be broad spectrum to inhibit the survival of any intracellular pathogens (or subgroup thereof, e.g., bacteria, e.g., vacuolar intracellular), such as when it acts on host or pathogen function that is critical for the ability of the pathogen to infect, survive or replicate within its host.
  • pathogens or subgroup thereof, e.g., bacteria, e.g., vacuolar intracellular
  • the agent thus identified is specific to the pathogen (at the level or the genus, species or strain) on which it was selected.
  • the method relies on the expression of a reporter molecule which is induced to be expressed from the pathogen only when it has invaded the host cell. At the extracellular environment its expression is generally null.
  • the pathogen is transfected or transformed with a plasmid that comprises a nucleic acid sequence encoding a reporter molecule under the regulation of a cis acting regulatory element active only in the intracellular environment.
  • the cis-acting regulatory element can be for example a promoter.
  • This for example a promoter of a gene that is induced intracellularly.
  • genes include but are not limited to the Salmonella pathogenicity island 2 genes (.s.saR, ssrB, SifA, sseA etc.) and the PhoPQ regulon (phop, pagC, mgtA etc).
  • Salmonella pathogenicity island 2 genes .s.saR, ssrB, SifA, sseA etc.
  • PhoPQ regulon phop, pagC, mgtA etc.
  • phop Listeria monocytogenes prfA, actA, hly, plcA, plcB and others.
  • examples include CAP10, FKS1, S0D1 and S0D2.
  • examples include CAP10, FKS1, S0D1 and S0D2.
  • the cbpl and Yps3 are induced intracellularly.
  • the var and PfEMPl genes are induced during host cell infection.
  • the rep (replication) and cap (capsid) genes are induced during cell infection and can be used for the construction of the reporter system.
  • the promoter is the ssek3 promoter (see e.g., Brown et al. 2011 PEGS ONE 6(3):el7824).
  • the reporter gene can be for example any that emits a measurable or detectable signal such as a bioluminescent or fluorescent signal: fluorescent proteins (e.g., green/red/yellow fluorescent protein), lacZ (P-galactosidase), luciferase (lux, e.g., luxCDABE) as described in the Examples section which follows, CAT (Chloramphenicol Acetyltransferase) and GUS (P-glucuronidase).
  • fluorescent proteins e.g., green/red/yellow fluorescent protein
  • lacZ P-galactosidase
  • luciferase lux, e.g., luxCDABE
  • CAT Chloramphenicol Acetyltransferase
  • GUS P-glucuronidase
  • Enhancer elements can stimulate transcription up to 1,000 fold from linked homologous or heterologous promoters. Enhancers are active when placed downstream or upstream from the transcription initiation site. These can be derived from the above genes as well.
  • the promoter is preferably positioned approximately the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.
  • Polyadenylation sequences can also be added to the expression vector in order to increase the efficiency of translation. Two distinct sequence elements are required for accurate and efficient poly adenylation: GU or U rich sequences located downstream from the polyadenylation site and a highly conserved sequence of six nucleotides, AAUAAA, located 11-30 nucleotides upstream. Termination and polyadenylation signals that are suitable for some embodiments of the invention include those derived from SV40.
  • the expression vector of some embodiments of the invention may typically contain other specialized elements intended to increase the level of expression of cloned nucleic acids or to facilitate the identification of cells that carry the recombinant DNA.
  • a number of animal viruses contain DNA sequences that promote the extra chromosomal replication of the viral genome in permissive cell types. Plasmids bearing these viral replicons are replicated episomally as long as the appropriate factors are provided by genes either carried on the plasmid or with the genome of the host cell.
  • the vector may or may not include a eukaryotic replicon
  • the expression vector of some embodiments of the invention can further include additional polynucleotide sequences that allow, for example, the translation of several proteins from a single mRNA such as an internal ribosome entry site (IRES) and sequences for genomic integration of the promoter-chimeric polypeptide.
  • IRS internal ribosome entry site
  • the individual elements comprised in the expression vector can be arranged in a variety of configurations.
  • enhancer elements, promoters and the like, and even the polynucleotide sequence(s) can be arranged in a "head-to-tail" configuration, may be present as an inverted complement, or in a complementary configuration, as an anti-parallel strand. While such variety of configuration is more likely to occur with non-coding elements of the expression vector, alternative configurations of the coding sequence within the expression vector are also envisioned.
  • Non-limiting examples of bacterial constructs include the pET series of E. coli expression vectors [Studier et al. (1990) Methods in Enzymol. 185:60-89) or pCS26 [
  • the pathogen may be transformed stably or transiently with the nucleic acid constructs disclosed herein.
  • stable transformation the nucleic acid molecule is integrated into the cell genome and as such it represents a stable and inherited trait.
  • transient transformation the nucleic acid molecule is expressed by the cell transformed but it is not integrated into the genome and as such it represents a transient trait.
  • the transformed pathogen i.e., genetically modified pathogen
  • the host organism can be plant or animal host (e.g., human, see Examples section).
  • the transformed cells are obtained, and the presence of the genetically modified pathogen therein is affirmed (by detecting the signal from the reporter molecule), it is subjected to treatment with the test agent.
  • antibiotics which acts extracellularly is added to the culture to eliminate extracellular pathogens. This allows measuring the effect of the examined agent on the invasion/survival/ replication of the intracellular pathogen.
  • the reporter activity is measured in comparison to a control sample which does not include the test agent.
  • a decrease in the reporter activity in the presence of the agent in comparison to an absence thereof is indicative that the agent can be used against intracellular pathogens.
  • the assay can be done in large scale, testing many agents individually or in a pooled manner, in which different compounds are tested together.
  • the method further comprises determining viability of the host cells in the presence of the agent in comparison to an absence thereof, and wherein substantially the same viability (e.g., about 5-10 %, 5-20 %, 5-15 %) is indicative that the agent is not-toxic to host cells.
  • substantially the same viability e.g., about 5-10 %, 5-20 %, 5-15 % is indicative that the agent is not-toxic to host cells.
  • any of the methods described herein or uses of the compounds or agents may be performed in vitro, e.g., in cell models (e,g., as described in the Examples section which follows) or on surfaces, such as described hereinbelow.
  • any of the methods described herein or uses of the compounds or agents may be performed in vivo (necessitating their administration to a human or animal host).
  • the compounds or agents described herein and/or derivative thereof may be administered per se, or as part of a pharmaceutical composition.
  • pharmaceutical composition refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients.
  • the purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
  • active ingredient refers to the agents of the present invention accountable for the intended biological effect.
  • physiologically acceptable carrier and “pharmaceutically acceptable carrier”, which may be used interchangeably, refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
  • An adjuvant is included under these phrases.
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.
  • excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.
  • agents of the present invention can be provided to the individual with additional active agents to achieve an improved therapeutic effect as compared to treatment with each agent by itself.
  • compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, drageemaking, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the active ingredients of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient.
  • Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
  • the compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • compositions described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative.
  • the compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
  • a suitable vehicle e.g., sterile, pyrogen-free water based solution
  • the preparation of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
  • the preparation of the present invention may also be formulated as topical compositions, such as a spray, a cream, a mouthwash, a wipe, a foam, a soap, an oil, a solution, a lotion, an ointment, a paste and a gel.
  • topical compositions such as a spray, a cream, a mouthwash, a wipe, a foam, a soap, an oil, a solution, a lotion, an ointment, a paste and a gel.
  • compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated.
  • the therapeutically effective amount or dose can be estimated initially from in vitro assays.
  • a dose can be formulated in animal models and such information can be used to more accurately determine useful doses in humans.
  • Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals.
  • the data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage may vary depending upon the dosage form employed and the route of administration utilized.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. [See e.g., Fingl, et al., (1975) "The Pharmacological Basis of Therapeutics", Ch. 1 p.l].
  • dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
  • compositions to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
  • the compound is represented by Formula I:
  • n is 0, 1, 2, 3, 4, or a higher integer, preferably, 0, 1, or 2, and more preferably is 0 or 1;
  • R 1 and R 2 are each independently selected from hydrogen and alkyl
  • R 3 -R 14 are each independently selected from hydrogen, alkyl, cycloalkyl, halo, haloalkyl, hydroxy, alkoxy, thiol, thioalkoxy, amine, cyano and nitro, and from any of the other substituents as described herein;
  • R 15 -R 24 are each independently selected from hydrogen, alkyl, cycloalkyl, amine, halo, hydroxy, thiol, alkoxy, and thioalkoxy;
  • R 25 is selected from hydrogen, alkyl and cycloalkyl.
  • n is 1.
  • R 9 and R 10 are absent.
  • R 9 and R 10 in each CR 9 R 10 moiety can be the same or different.
  • R 9 and R 10 when present, are each hydrogen.
  • At least one, and preferably each, of R 1 and R 2 is alkyl, and preferably a lower alkyl of, e.g., 1 to 6, or 1 to 4 carbon atoms in length.
  • the alkyl can be linear or branched and can be substituted or substituted.
  • the alkyl is unsubstituted.
  • the alkyl is a linear unsubstituted lower alkyl as described herein.
  • At least one, and preferably each, of R 1 and R 2 is alkyl, and the total number of carbon atoms in R 1 and R 2 is from 2 to 6, or from 2 to 4, and is, for example, 2, 3, or 4.
  • R 1 and R 2 are each independently selected from hydrogen, methyl, ethyl, propyl and isopropyl.
  • R 1 and R 2 are each methyl; or R 1 is methyl and R 2 is ethyl; or Ri is isopropyl and R 2 is hydrogen.
  • R 3 -R 8 are each hydrogen; such that the tetrahydropyrane ring in Formula I is substituted solely at the position adjacent to the oxygen (when one or both of R 1 and R 2 is/are an alkyl as described herein).
  • the tetrahydropyrane ring is substituted at one or more other positions, such that one or more of R 3 -R 8 is other than hydrogen.
  • R 3 -R 13 are each hydrogen.
  • R 9 -R 13 are each hydrogen.
  • R 11 -R 13 are each hydrogen.
  • one or more of R 3 -R 13 is other than hydrogen, and can be, for example, alkyl, cycloalkyl, halo, haloalkyl, hydroxy, alkoxy, thiol, thioalkoxy, amine, cyano and nitro, or any of the other substituents as described herein.
  • R 11 and R 12 are each hydrogen, and at least one of R 13 and R 14 is other than hydrogen, and can be, for example, alkyl, cycloalkyl, halo, haloalkyl, hydroxy, alkoxy, thiol, thioalkoxy, amine, cyano and nitro, or any of the other substituents as described herein.
  • one or more of R 3 -R 13 is alkyl, preferably a lower alkyl, of from 1 to 6, or from 1 to 4 carbon atoms in length, preferably a lower unsubstituted alkyl, which can be linear or branched and is preferably linear.
  • R 14 is alkyl, preferably a lower alkyl, of from 1 to 6, or from 1 to 4 carbon atoms in length, preferably a lower unsubstituted alkyl, which can be linear or branched and is preferably linear (e.g., methyl, ethyl, propyl).
  • R 3 -R 13 are each hydrogen.
  • R 14 is methyl.
  • R 3 -R 13 are each hydrogen.
  • R 14 is other than hydrogen, the carbon to which Ru is attached features an asymmetric stereoconfiguration.
  • Embodiments of the present invention encompass both the S- and R- stereoconfigurations.
  • each of R 15 - R 19 is hydrogen.
  • At least one of R 15 -R 19 is an electron-donating substituent, for example, fluoro, alkyl, cycloalkyl, hydroxy, alkoxy, aryloxy, amine, thiol, thioalkoxy, thioaryloxy, and like substituents.
  • one or more of the substituents at the ortho and/or para position(s) with respect to the tetrahydropyrane substituent that is, one or more of R 15 , R 17 or R 19 is an electron donating substituent or is selected from fluoro, alkyl, cycloalkyl, hydroxy, amine, alkoxy, aryloxy, thiol, thioalkoxy, thioaryloxy, and like substituents.
  • R 15 -R 19 when at least one of R 15 -R 19 is an electron-donating substituent, as described herein, the substituent is “aprotic”, such that it is other than hydroxy, thiol or primary amine, or is other than hydroxy, thiol, primary amine, or secondary amine.
  • one or more of R 15 , R 17 or R 19 is alkyl (preferably a lower alkyl as described herein), fluoro, thioalkoxy (preferably lower thioalkoxy) or alkoxy (preferably a lower alkoxy, or from 1 to 6, or from 1 to 4 carbon atoms in length).
  • lower alkoxy or “lower thioalkoxy” it is meant that the alkyl in such a group is a lower alkyl as described herein in any of the respective embodiments and any combination thereof.
  • the alkyl is a linear alkyl. In some embodiments the alkyl is unsubstituted.
  • R 15 is alkoxy (preferably lower alkoxy such as methoxy or ethoxy or propoxy or isopropoxy or butoxy). According to some embodiments of any of the embodiments described herein, R 15 is methoxy.
  • each of R 20 - R 24 is hydrogen.
  • At least one of R 20 -R 24 is an electron-donating substituent, for example, fluoro, alkyl, cycloalkyl, hydroxy, alkoxy, aryloxy, amine, thiol, thioalkoxy, thioaryloxy, and like substituents.
  • one or more of the substituents at the ortho and/or para position(s) with respect to the attachment position is an electron donating substituent or is selected from fluoro, alkyl, cycloalkyl, hydroxy, amine, alkoxy, aryloxy, thiol, thioalkoxy, thioaryloxy, and like substituents.
  • R 20 - R 24 when at least one of R 20 - R 24 is an electron-donating substituent, as described herein, the substituent is “aprotic”, such that it is other than hydroxy, thiol or primary amine, or is other than hydroxy, thiol, primary amine, or secondary amine.
  • one or more of R 20 -R 24 or one or more of R 20 , R 22 or R 24 is fluoro, amine (preferably a tertiary amine), or alkoxy (preferably a lower alkoxy, as described herein).
  • R 22 is amine (preferably a tertiary amine), fluoro or alkoxy (preferably lower alkoxy).
  • R 22 is amine (preferably a tertiary amine).
  • the tertiary amine is preferably -NR’R”, with each of R’ and R” being independently alkyl or cycloalkyl, preferably each is alkyl, which can be the same or different.
  • the alkyl is a lower alkyl as described herein, for example methyl.
  • one or more of R 20 -R 24 or one or more of R 20 , R 22 or R 24 , or R 22 is dialkyl amine, for example, dimethylamine.
  • an exemplary compound of Formula I is: -1013431 (also referred to herein as C4).
  • exemplary compounds of Formula I include:
  • exemplary compounds of Formula I include:
  • the compound is represented by Formula II: wherein:
  • R 30 and R 31 are each independently selected from hydrogen, alkyl and cycloalkyl
  • R 32 -R 36 are each independently selected from hydrogen, alkyl, cycloalkyl, halo, haloalkyl, hydroxy, alkoxy, thiol, thioalkoxy, amine, cyano and nitro, provided that at least one of R 32 -R 36 , preferably at least one of R 32 , R 34 and R 36 is halo; and
  • A is selected from: wherein the curved line represents an attachment point to NR 30 -; and R 37 and R 38 are each independently selected from hydrogen alkyl and cycloalkyl.
  • R 30 and R 31 are each independently selected from hydrogen and alkyl.
  • the alkyl when one or each of R 30 and R 31 is alkyl, the alkyl is a lower alkyl as described herein.
  • R 30 and R 31 are each hydrogen.
  • one or more of R 32 -R 36 is halo and the remaining variables are each hydrogen.
  • one or more of the remaining variables is alkyl (preferably lower alkyl), cycloalkyl, haloalkyl, hydroxy, alkoxy, thiol, thioalkoxy, amine, cyano and nitro, preferably alkyl, or haloalkyl (e.g., CF3).
  • the one or more halo substituents can be chloro, bromo and/or iodo, or chloro and/or bromo, or chloro.
  • R 32 , R 34 and R 36 is halo, and according to some embodiments, one or more of R 32 , R 34 and R 36 is chloro. According to some embodiments of any of the embodiments of Formula II, one or more of
  • R 32 , R 34 and R 36 is halo (e.g., chloro), and each of R 30 and R 31 is hydrogen. According to some of these embodiments, one or more of R 32 -R 36 which is not halo are each hydrogen.
  • A is According to some embodiments of any of the embodiments described herein, A is according to some of these embodiments, at least two of R 32 -R 36 , preferably at least two of R 32 , R 34 and R 36 , are each halo, preferably chloro. According to some of these embodiments, one or more of R 32 -R 36 which is not halo are each hydrogen. According to some of these embodiments, each of R 30 and R 31 is hydrogen.
  • A is two of R 32 , R 34 and R 36 , are each halo, preferably chloro and the third is hydrogen, and R33 and R35 are each hydrogen. According to some of these embodiments, each of
  • R 30 and R 31 is hydrogen.
  • A is and no more than one of R 32 -R 36 , preferably one of R 32 , R 34 and R 36 , is halo, preferably chloro.
  • one or more of R 32 -R 36 which is not halo are each hydrogen.
  • each of R 30 and R 31 is hydrogen.
  • A is , R 32 is halo, preferably chloro, and R 33 -R 36 are each hydrogen. According to some of these embodiments, each of R 30 and R 31 is hydrogen.
  • R 37 and R38 are each independently selected from hydrogen and alkyl.
  • A is one or more of R 37 and R38 is alkyl, preferably a lower alkyl (e.g., methyl).
  • R 37 is alkyl (e.g., methyl).
  • A is R 37 and R38 are each hydrogen.
  • A can be a heteroaryl other than isoxazole, and can be, for example, furane, pyrazole, triazole, diazole, oxadiazole, pyrrole, and other heteroaryls of 5, 6, 7-membered ring and one, two or more heteroatoms selected from nitrogen and oxygen.
  • the compound is: (C7; PCM 0001349; Compound 5) or (Compound 6).
  • the compound is represented by Formula III: wherein: R 40 -R 44 are each independently selected from hydrogen, alkyl, cycloalkyl, halo, hydroxy, alkoxy, thiol, thioalkoxy, amine; and
  • R 45 -R 52 are each independently selected from hydrogen, alkyl, cycloalkyl, halo, hydroxy, alkoxy, thiol, thioalkoxy, and amine.
  • At least one of R 40 -R 44 is other than hydrogen, and in some embodiments, one or more of R 40 -R 44 , is alkyl, cycloalkyl, halo, alkoxy, thioalkoxy, or amine (e.g., a tertiary amine).
  • At least one of R 40 -R 44 is cycloalkyl.
  • one of R 40 - R 44 is cycloalkyl.
  • R 40 is other than hydrogen and is alkyl, cycloalkyl, halo, alkoxy, thioalkoxy, or amine (e.g., a tertiary amine).
  • R 40 is cycloalkyl
  • the cycloalkyl is cyclopentyl, cyclobutyl, cyclopropyl, cyclohexyl, cycloheptyl or cyclooctyl.
  • the cycloalkyl is cyclopentyl or cyclohexyl.
  • R 40 is a cycloalkyl and is preferably cyclopentyl or cyclohexyl.
  • R 40 -R 44 when one or more of R 40 -R 44 is other than hydrogen, the remaining variables can be each hydrogen, or, one or more can be other than hydrogen.
  • R 40 is other than hydrogen (and is, for example, cycloalkyl) and R 41 -R 44 are each hydrogen.
  • R 40 is a cycloalkyl and at least one of R 41 -R 44 , is other than hydrogen, and in some embodiments, one or more of R 41 -R 44 , is alkyl, cycloalkyl, halo, alkoxy, thioalkoxy, or amine (e.g., a tertiary amine).
  • each of R 45 - R 50 is hydrogen.
  • At least one of R 45 -R 50 is other than hydrogen, and is selected from hydroxy, thiol, alkoxy and thioalkoxy. According to some embodiments of any of the embodiments described herein, at least one of R 45 -R 50 is hydroxy.
  • R 47 is hydroxy.
  • R 45 , R 46 and R 48 -R 50 are each hydrogen.
  • one or more of R 45 , R 46 and R 48 -R 50 can be, for example, alkyl (preferably lower alkyl), halo, hydroxy, thiol, amine, alkoxy, thioalkoxy or any of the other substituents as described herein.
  • R 47 is thiol.
  • R 45 , R 46 and R 48 -R 50 are each hydrogen.
  • one or more of R 45 , R 46 and R 48 -R 50 can be, for example, alkyl (preferably lower alkyl), halo, hydroxy, thiol, amine, alkoxy, thioalkoxy or any of the other substituents as described herein.
  • R 45 is hydroxy or thiol.
  • R 46 -R 50 are each hydrogen.
  • one or more of R 46 -R 50 can be, for example, alkyl (preferably lower alkyl), halo, hydroxy, thiol, amine, alkoxy, thioalkoxy or any of the other substituents as described herein.
  • R 50 is hydroxy or thiol.
  • R 45 -R 49 are each hydrogen.
  • one or more of R 45 -R 49 can be, for example, alkyl (preferably lower alkyl), halo, hydroxy, thiol, amine, alkoxy, thioalkoxy or any of the other substituents as described herein.
  • each of R 51 and R 52 is hydrogen, such that the amine is a primary amine.
  • one or each of R 51 and R 52 is other than hydrogen, such that the amine or secondary or tertiary amine.
  • R 52 is other than hydrogen and in some embodiments it is an alkyl.
  • the alkyl is a lower alkyl, as described herein, which can be linear or branched, and is preferably unsubstituted.
  • the alkyl is a lower branched (e.g., bulky) alkyl, such as isopropyl, isobutyl, tert-butyl, isopentyl, amyl, etc.
  • R 52 is tert-butyl.
  • R 51 is hydrogen.
  • the compound is: also referred to herein as C2.
  • the compound is represented by Formula IV :
  • R 61 -R 64 are each independently selected from hydrogen, alkyl, cycloalkyl, halo, haloalkyl, amine, hydroxy, alkoxy, thiol, thioalkoxy, or, alternatively, two of R 61 -R 64 form together a cyclic ring, which can be alicyclic, heteroalicyclic, aryl or heteroaryl, as these are defined herein;
  • R 65 and R66 are each independently selected from hydrogen, alkyl and cycloalkyl
  • R 67 -R 78 are each independently selected from hydrogen, alkyl, cycloalkyl, halo, haloalkyl, amine, hydroxy, alkoxy, thiol and thioalkoxy;
  • R 80 represents one or more (1, 2, 3, 4 or 5) substituents on the phenyl ring, or is absent, wherein each of these substituents, if present, is independently selected from alkyl, cycloalkyl, halo, haloalkyl, amine, hydroxy, alkoxy, thiol, and thioalkoxy.
  • one or each of R 65 and R 66 is selected from hydrogen and alkyl.
  • each of R 65 and Ree is hydrogen.
  • one or more of R 61 -R 64 is other than hydrogen. According to some embodiments of any of the embodiments described herein, one or more of R 61 -R 64 is selected from alkyl, haloalkyl and halo, and is preferably a haloalkyl such as trihaloalkyl (e.g., CF3).
  • R 61 -R 64 when one or more of R 61 -R 64 is other than hydrogen, the others can be hydrogen or any of the other substituents.
  • one or more of R 61 -R 64 is selected from alkyl, haloalkyl and halo and is preferably a haloalkyl such as trihaloalkyl (e.g., CF3), and the others can be hydrogen or any of the other substituents.
  • R 64 is other than hydrogen.
  • R 64 is selected from alkyl, haloalkyl and halo, and is preferably a haloalkyl such as trihaloalkyl (e.g., CF 3 ).
  • R 64 is other than hydrogen and is alkyl, haloalkyl or halo, and is preferably trihaloalkyl as described herein
  • R 61 -R 63 are each hydrogen, or one or more is other than hydrogen, and can be any of the other substituents as described herein.
  • R 67 -R 70 are each hydrogen.
  • one or more of R 67 -R 70 is other than hydrogen, and can be, for example, hydroxy, thiol, amine, halo, alkyl, alkoxy, thioalkoxy, haloalkyl, or any of the other substituents as described herein.
  • one or more of R 67 -R 70 is/are hydroxy.
  • R 71 -R 76 are each hydrogen.
  • one or more of R 71 -R 76 is other than hydrogen, and can be, for example, hydroxy, thiol, amine, halo, alkyl, alkoxy, thioalkoxy, haloalkyl, or any of the other substituents as described herein.
  • one or more of R 71 -R 76 is/are hydroxy.
  • At least one of R 71 -R 76 is hydroxy.
  • R 71 -R 73 , R 75 and R 76 are each hydrogen and R 74 is hydroxy.
  • R 77 and R 78 are each hydrogen.
  • one or more is alkyl, for example, a lower alkyl as described herein.
  • R 79 is absent.
  • the respective phenyl ring is substituted by one or more substituents, as indicated.
  • the compound is: and is also referred to herein as C3.
  • the compound is represented by Formula V :
  • R 80 and R 81 are each independently selected from hydrogen, alkyl and cycloalkyl
  • R 82 -R 86 are each independently selected from hydrogen, alkyl, cycloalkyl, halo, haloalkyl, amine, hydroxy, alkoxy, thiol, and thioalkoxy;
  • R 87 -R 90 are each independently selected from hydrogen, alkyl, cycloalkyl, halo, haloalkyl, amine, hydroxy, alkoxy, thiol, and thioalkoxy;
  • R 91 -R 94 are each independently selected from hydrogen, alkyl, cycloalkyl, halo, haloalkyl, amine, hydroxy, alkoxy, thiol, and thioalkoxy;
  • R 95 -R 98 are each independently selected from hydrogen, alkyl, cycloalkyl, halo, haloalkyl, amine, hydroxy, alkoxy, thiol, and thioalkoxy.
  • R 80 and R 81 are each independently selected from alkyl and hydrogen, and according to some embodiments, each is hydrogen.
  • R 82 and R 83 are each independently selected from alkyl (e.g., a lower alkyl) and hydrogen, and according to some embodiments, each is hydrogen.
  • At least one of R 84 -R 86 is other than hydrogen, and is some embodiments, at least one of R 84 -R 86 is hydroxy, thiol, alkoxy, thioalkoxy or amine. According to some of these embodiments, at least one of R 84 -R 86 is hydroxy. According to some of any of the embodiments described herein, R 84 is other than hydrogen, and in some embodiments it is hydroxy, thiol, alkoxy, thioalkoxy or amine. According to some of any of the embodiments described herein, R 84 is hydroxy.
  • R 84 -R 86 when one or more of R 84 -R 86 is other than hydrogen, the other variables can be hydrogen, or any other substituent, for example, alkyl, haloalkyl, halo, etc.
  • R 84 is hydroxy and R 85 and R 86 are each hydrogen.
  • each of R 87 - R 90 is hydrogen.
  • At least one of R 87 -R 90 is other than hydrogen, and can be, for example, hydroxy, thiol, amine, thioalkoxy, and/or alkoxy.
  • the remaining variables can be hydrogen, or, for example, alkyl, haloalkyl, halo and/or cycloalkyl.
  • one or more of R 87 -R 90 is hydroxy.
  • R 87 is hydroxy
  • R 88 -R 90 are each hydrogen.
  • one or more of R 88 -R 90 is other than hydrogen and is a substituent as described herein.
  • R 90 is hydroxy, and R 87 -R 89 are each hydrogen.
  • R 87 -R89 is other than hydrogen and is a substituent as described herein.
  • each of R91- R94 is hydrogen.
  • one or more of R 91 -R 94 is other than hydrogen, and can be, independently, for example, alkyl, cycloalkyl, haloalkyl, halo, and like substituents.
  • each of R 95 - R 98 is hydrogen.
  • one or more of R 95 -R 98 is other than hydrogen and is independently selected from the substituents as described herein.
  • one or more of R 95 -R 98 is alkyl, preferably a lower alkyl.
  • the others are each hydrogen, or one or more can be a substituent other than hydrogen as described herein.
  • two or more of R 95 -R 98 is alkyl, preferably a lower alkyl.
  • the others are each hydrogen, or one or more can be a substituent other than hydrogen as described herein.
  • one or more, or each, of R 96 and R 97 is an alkyl, preferably a lower alkyl.
  • R 96 and R 97 is alkyl, it can be the same or different.
  • the remaining R 95 and R 98 are each hydrogen.
  • the compound is:
  • the compound is represented by Formula VI:
  • Rioo is hydrogen, alkyl or cycloalkyl
  • R 101 -R 105 are each independently selected from hydrogen, alkyl, cycloalkyl, aryl, halo, haloalkyl, amine, hydroxy, alkoxy, thiol, thioalkoxy, heteroaryl, cyano, and nitro;
  • R106-R109 are each independently selected from hydrogen, alkyl, cycloalkyl, halo, haloalkyl, amine, hydroxy, alkoxy, thiol and thioalkoxy;
  • R 110 -R 113 are each independently selected from hydrogen, alkyl, cycloalkyl, halo, haloalkyl, amine, hydroxy, alkoxy, thiol and thioalkoxy, or, alternatively, two of R 110 -R 113 form together a substituted or unsubstituted cyclic ring (alicyclic, heteroalicyclic, aryl or heteroaryl); and
  • R 114 -R 117 are each independently selected from hydrogen, alkyl, cycloalkyl, halo, haloalkyl, amine, hydroxy, alkoxy, thiol and thioalkoxy.
  • Rioo is hydrogen or alkyl, and is preferably hydrogen.
  • At least one of R 101 -R 105 is other than hydrogen and can be, for example, alkyl, cycloalkyl, haloalkyl or aryl.
  • At least one of R 101 -R 105 is aryl, preferably phenyl.
  • R 101 is other than hydrogen and can be, for example, alkyl, cycloalkyl, haloalkyl or aryl.
  • R 101 is aryl (e.g., phenyl).
  • R 102 -R 105 are each hydrogen.
  • one or more of R 102 -R 105 is other than hydrogen and each independently can be a substituent as described herein.
  • R 113 form together a substituted or unsubstituted cyclic ring, which can be alicyclic, aryl, heteroalicyclic or heteroaryl, as these terms are defined herein, wherein each can be substituted or substituted, as defined herein. According to some embodiments of any of the embodiments described herein, two of R 110 -R 113 form together a substituted or unsubstituted heteroaryl, for example, a 5-membered, or six-membered heteroaryl.
  • R 112 and R 113 form together a substituted or unsubstituted cyclic ring, which can be alicyclic, aryl, heteroalicyclic or heteroaryl, as these terms are defined herein, wherein each can be substituted or substituted, as defined herein.
  • R112 and R 113 form together a substituted or unsubstituted heteroaryl, for example, a 5-membered, or six-membered heteroaryl.
  • R 112 and R 113 form together a substituted or unsubstituted imidazole, for example, 2-mcthyl-2/7-imidazolc.
  • R 110 and R 111 are each hydrogen.
  • one or each of R 110 and Rm can be other than hydrogen, as described herein, for example, can independently be alkyl, haloalkyl, and cycloalkyl, etc.
  • R106-R109 are each hydrogen and/or R 114 -R 117 are each hydrogen.
  • one or more of these variables can be other than hydrogen, and can be, for example, alkyl, cycloalkyl, halo, haloalkyl, hydroxy, alkoxy, and/or any other substituent as described herein.
  • the compound is: also referred to herein as C6.
  • the compound is represented by Formula VII: wherein:
  • X is O, S or NR 120 ;
  • R 119 and R 120 are each independently selected from hydrogen, alkyl or cycloalkyl;
  • R 121 -R 125 are each independently selected from hydrogen, alkyl, cycloalkyl, aryl, halo, haloalkyl, amine, hydroxy, alkoxy, aryloxy, thioaryloxy, thiol, thioalkoxy, heteroaryl, cyano and nitro, or, alternatively or in addition, two of R 121 -R 125 form together a substituted or unsubstituted cyclic ring (alicyclic, heteroalicyclic, aryl or heteroaryl); and
  • R 126 -R 129 are each independently selected from hydrogen, alkyl, cycloalkyl, halo, haloalkyl, amine, hydroxy, alkoxy, thiol, thioalkoxy, heteroalicyclic, heteroaryl, and aryloxy.
  • X is NR 120 .
  • R 119 and R 120 are each independently selected from hydrogen and alkyl.
  • R 119 and R 120 are each hydrogen.
  • At least one of R 121 -R 125 is other than hydrogen.
  • At least one of R 126 -R 129 is other than hydrogen.
  • At least one of R 121 -R 125 and at least one of R 126 -R 129 is other than hydrogen.
  • At least one of R 121 -R 125 is alkoxy or aryloxy.
  • At least one of R 126 -R 129 is alkoxy, aryloxy or a heterocyclic, for example, a heteroalicyclic or a heteroaryl.
  • R 126 -R 129 is a heteroaryl, the remaining of these variables are each hydrogen.
  • none of R 126 -R 129 per se (alone) is a heteroaryl.
  • none of R 126 - R 129 per se (alone) is a heterocyclic ring, as described herein.
  • two of R 121 - R 125 form together a cyclic ring, which is preferably an alicyclic ring or a heteroalicyclic ring.
  • two of R 121 - R 125 form together a heteroalicyclic ring.
  • the heteroalicyclic ring can be, for example, a 5- membered or 6-membered ring.
  • the heteroalicyclic ring can include one or more heteroatoms, for example, one or more of O, N and S.
  • the heteroalicyclic ring comprises one or two oxygen atoms, and can be, for example, tetrahydrofurane, tetrahydropyrane, dioxolane, dioxane, and like cyclic moieties.
  • R 121 and R 122 form together a cyclic ring as described herein in any of the respective embodiments, for example, dioxolane.
  • two of R 121 - R 125 form together a cyclic ring, the remaining variables can each be hydrogen or independently one or more of the other substituents as described herein, for example, alkyl, halo, haloalkyl, etc.
  • At least two of R 126 -R 129 are each independently an alkoxy.
  • At least one, and preferably at least two, of R 126 -R 129 are each independently an alkoxy, a thioalkoxy, thiol or hydroxy.
  • the remaining variables are each hydrogen, or, alternatively, one or more is other than hydrogen and can be a substituent as described herein.
  • At least one, and preferably at least two, of R 126 -R 129 are each independently an alkoxy.
  • one or each of R 126 and R 128 is independently an alkoxy, a thioalkoxy, thiol or hydroxy.
  • the remaining variables are each hydrogen, or, alternatively, one or more is other than hydrogen and can be a substituent as described herein.
  • R 126 and R 128 are each independently an alkoxy, and the compound is represented by Formula Vila:
  • R 130 and R 131 are each independently selected from alkyl, cycloalkyl, and heteroalicyclic and the other variables are as defined herein in any of the respective embodiments of Formula VII and any combination thereof.
  • R 121 and R 122 form together the cyclic ring, preferably the heteroalicyclic ring, as described herein in any of the respective embodiments and any combination thereof.
  • R 121 and R 122 form together a dioxolane.
  • At least one of R 123 -R 125 , preferably R 125 is halo, preferably chloro.
  • R 130 and R 131 each independently is or comprise a heteroalicyclic.
  • R 130 is a heteroalicyclic, as defined herein, which can be, for example, a 5-membered or 6-membered ring comprising one or more of O, S and N.
  • R 130 is a 5-membered or 6- membered ring comprising one or more O atoms, and can be, for example, tetrahydrofurane, tetrahydropyrane, dioxolane, and like cyclic moieties.
  • R 130 is tetrahydropyrane.
  • R 131 is an alkyl substituted by a heteroalicylic.
  • the alkyl is a lower alkyl, as described herein, for example, methylene, ethylene or propylene, which is substituted by or terminated by a heteroalicyclic.
  • the heteroalicyclic is a 5- membered or 6-membered ring comprising one or more of O, S and N.
  • R 130 is a 5-membered or 6-membered ring comprising one or more N atoms, and can be, for example, piperidine, piperazine, oxazolidine, imidazolidine, dihydropyrimidine, and like moieties.
  • the heteroalicyclic ring is piperazine.
  • R 131 is ethylpiperazine.
  • the compound is:
  • PCM-0094889 and is also referred to herein as C 1.
  • R 130 is a heteroalicyclic and R 131 is an alkyl, as described herein in any of the respective embodiments, substituted or terminated by a heteroaryl.
  • At least one of R 121 -R 125 is aryloxy, preferably hetero aryloxy.
  • the remaining variables can be each hydrogen, or can be independently a substituent as described herein.
  • R 123 is the aryloxy, for example, a heteroaryloxy.
  • R 121 , R 122 , R 124 and R 125 are each hydrogen.
  • one or more of R 121 , R 122 , R 124 and R 125 is other than hydrogen and can be, for example, any of the substituents as described herein, for example, alkyl (e.g., lower alkyl).
  • At least one of R 126 -R 129 is a heteroaryl, which can be, for example, a 5-membered or 6- membered ring that comprises one or more O, N and/or S atoms.
  • at least one of R 126 -R 129 is a 5-membered or 6-membered ring that comprises one or more O atoms, for example, furyl or pyranyl.
  • at least one of R 126 -R 129 is furyl, more preferably a substituted furyl.
  • R 127 is the heteroaryl.
  • R 127 is furyl, and the compound is represented by Formula Vllb:
  • R 132 -R 134 are each independently selected from hydrogen, alkyl, cycloalkyl, halo, haloalkyl, amine, heteroalicyclic, heteroaryl, cyano and nitro, and the other variables are described herein for Formula VII, in any of the respective embodiments and any combination thereof.
  • At least one of R 121 -R 125 is an aryloxy, preferably a heteroaryloxy, as described herein in any of the respective embodiments.
  • R 123 is a heteroaryloxy, preferably 7-triazolopyridinoxy.
  • each of R 121 , R 122 , R 124 and R 125 is hydrogen.
  • at least one of R 121 , R 122 , R 124 and R 125 , preferably R 124 is alkyl, preferably methyl.
  • At least one of R 132 -R 134 is an alkyl, preferably a substituted alkyl.
  • the alkyl is substituted by a moiety that comprises an amine and/or SO 2 , preferably a hydrocarbon interrupted and/or substituted by an amine and/or SO 2 .
  • At least one of R 132 -R 134 is an alkyl substituted by amine.
  • the amine is substituted, that is, it is a secondary or tertiary amine.
  • the amine is substituted by at least one alkyl which is further substituted by a sulfone.
  • Vllb at least one of R 132 -R 134 , preferably R 132 , is an alkyl and the alkyl is -CH 2 -NH-CH 2 -CH 2 - SO 2 -CH3.
  • the compound is: , also referred to herein as C5.
  • R 123 is other than heteroaryloxy; and/or R 132 is other than an alkyl substituted by a moiety that comprises an amine and/or SO 2 , or a hydrocarbon interrupted and/or substituted by an amine and/or SO 2 .
  • the compound is one or more of the compounds presented in FIG. 2B.
  • the compound is one or more of the compounds presented in FIG. 10B.
  • linking moiety or “linking group” describes a group that connects two or more moieties or groups in a compound.
  • a linking moiety is typically derived from a bi- or tri-functional compound, and can be regarded as a bi- or tri-radical moiety, which is connected to two or three other moieties, via two or three atoms thereof, respectively.
  • linking moieties include a hydrocarbon moiety or chain, optionally interrupted by one or more heteroatoms, as defined herein, and/or any of the chemical groups listed below, when defined as linking groups.
  • end group When a chemical group is referred to herein as “end group” it is to be interpreted as a substituent, which is connected to another group via one atom thereof.
  • alkyl describes a saturated aliphatic hydrocarbon including straight chain and branched chain groups.
  • the alkyl group has 1 to 20 carbon atoms. Whenever a numerical range; e.g., "1-20", is stated herein, it implies that the group, in this case the alkyl group, may contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms.
  • the alkyl is a lower alkyl having 1 to 8, or 1 to 6, or 1 to 4 carbon atoms.
  • the alkyl group may be substituted or unsubstituted, as indicated herein.
  • the alkyl group can be an end group, as this phrase is defined hereinabove, wherein it is attached to a single adjacent atom, or a linking group, as this phrase is defined hereinabove, which connects two or more moieties via at least two carbons in its chain.
  • a linking group it is also referred to herein as “alkylene” or “alkylene chain”.
  • alkaryl describes an alkyl, as defined herein, which is substituted by one or more aryl or heteroaryl groups. An example of alkaryl is benzyl.
  • alkyl encompasses “alkaryl” unless specifically indicated otherwise.
  • alkenyl describes an alkyl, as defined herein, which contains a carbon-to-carbon double bond.
  • alkynyl describes an alkyl, as defined herein, which contains carbon-to-carbon triple bond.
  • cycloalkyl or “alicyclic” describes an all-carbon monocyclic or fused ring (i.e., rings which share an adjacent pair of carbon atoms) group where one or more of the rings does not have a completely conjugated pi-electron system.
  • the cycloalkyl group may be substituted or unsubstituted, as indicated herein.
  • aryl describes an all-carbon monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups having a completely conjugated pi-electron system.
  • the aryl group may be substituted or unsubstituted, as indicated herein.
  • the aryl group can be an end group, as this phrase is defined hereinabove, wherein it is attached to a single adjacent atom, or a linking group, as this phrase is defined hereinabove, which connects two or more moieties via at least two carbons in its chain.
  • arylene for example, phenylene.
  • heteroaryl describes a monocyclic or fused ring (i.e., rings which share an adjacent pair of atoms) group having in the ring(s) one or more atoms, such as, for example, nitrogen, oxygen and sulfur and, in addition, having a completely conjugated pi-electron system.
  • heteroaryl groups include pyrrole, furan, thiophene, imidazole, oxazole, thiazole, carbazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline and purine.
  • heteroalicyclic or “heterocyclyl” describes a monocyclic or fused ring group having in the ring(s) one or more atoms such as nitrogen, oxygen and sulfur.
  • the rings may also have one or more double bonds. However, the rings do not have a completely conjugated pi- electron system.
  • Representative examples are piperidine, piperazine, tetrahydrofuran, tetrahydropyrane, morpholino and the like.
  • alkoxy describes both an -O-alkyl, an -O-cycloalkyl and an -O-heteroalicyclic group, as defined herein.
  • aryloxy describes an -O-aryl and an -O-heteroaryl, as defined herein.
  • heteroaryloxy describes specifically -O-heteroaryl.
  • Each of the alkyl, cycloalkyl, heteroalicyclic, aryl and heteroaryl groups, including alkylene and arylene groups, in the general formulas herein, including when included in alkoxy, thioalkoxy, thioaryloxy, aryloxy, etc. may be substituted by one or more substituents, whereby each substituent group can independently be, for example, halogen, alkyl, alkoxy, cycloalkyl, alkoxy, cyano, nitro, amine, hydroxyl, thiol, thioalkoxy, thiohydroxy, carboxy, amide, aryl and aryloxy, depending on the substituted group and its position in the molecule.
  • substituents are also contemplated, for example, sulfinyl, sulfonyl, sulfonate, sulfate, azide, phosphonyl, phosphinyl, imine, hydrazone, carbonyl, thiocarbonyl, a urea group, a thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, S -thiocarbamyl, C- amido, N-amido, C-carboxy, O-carboxy, sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide.
  • halide describes fluorine, chlorine, bromine or iodine.
  • haloalkyl describes an alkyl group as defined herein, further substituted by one or more halide(s).
  • haloalkyl is encompassed by the term “alkyl”.
  • trihaloalkyl describes an alkyl, typically methyl, which is substituted by 3 halo atoms, which can be the same or different. Examples include CF3 and CCI3, although other trihaloalkyls are contemplated.
  • hydroxyl or "hydroxy” describes a -OH group.
  • thiohydroxy or “thiol” describes a -SH group.
  • thioalkoxy describes both an -S-alkyl group, and a -S-cycloalkyl group, as defined herein.
  • thioaryloxy describes both an -S-aryl and a -S-heteroaryl group, as defined herein.
  • amine each refer to either a -NR’R” group or a - N + R’R”R’ ’ ’ group, wherein R’ , R” and R’ ’ ’ are each hydrogen or a substituted or non-substituted alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic (linked to amine nitrogen via a ring carbon thereof), aryl, or heteroaryl (linked to amine nitrogen via a ring carbon thereof), as defined herein.
  • R’, R” and R”’ are hydrogen or alkyl comprising 1 to 4 carbon atoms.
  • R’ and R” are hydrogen.
  • the carbon atom of an R’, R” or R”’ hydrocarbon moiety which is bound to the nitrogen atom of the amine is not substituted by oxo (unless explicitly indicated otherwise), such that R’, R” and R’” are not (for example) carbonyl, C-carboxy or amide, as these groups are defined herein.
  • a "nitro” group refers to a -NO 2 group.
  • nitrogen-containing heteroaryls include, but are not limited to thiadiazole, pyridine, pyrrole, oxazole, indole, purine and the like. Other moieties are also contemplated.
  • nitrogen-containing heteroalicyclic include, but are not limited to, morpholine, thiomorpholine, piperidine, piperazine, hexahydro azepine and tetrahydropyrane. Other moieties are also contemplated.
  • piperazine refers to a group or a or a group, where R’ and R” are as defined hereinabove.
  • pyrrolidine refers to a group or a group, with R’ as defined herein.
  • pyridine refers to a group.
  • pyrrole refers to a group, with R’ as defined herein.
  • morpholine refers to a group, and encompasses also thiomorpholine.
  • thiomorpholine refers to a group.
  • hexahydro azepine refers to a group.
  • halo refers to fluorine, chlorine, bromine or iodine.
  • a “sulfonamide” or “sulfonamido” group encompasses both S-sulfonamido and N- sulfonamido groups, as defined herein.
  • amide or “amido” group encompasses C-amido and N-amido groups, as defined herein.
  • phosphinyl describes a -PR’R” group, with each of R’ and R” as defined hereinabove.
  • hydrazine describes a -NR’-NR”R’” group, with R’, R”, and R’” as defined herein.
  • the compound described herein may be in a form of a salt, for example, a pharmaceutically acceptable salt, and/or in a form of a prodrug.
  • the phrase “pharmaceutically acceptable salt” refers to a charged species of the parent compound and its counter-ion, which is typically used to modify the solubility characteristics of the parent compound and/or to reduce any significant irritation to an organism by the parent compound, while not abrogating the biological activity and properties of the administered compound.
  • a pharmaceutically acceptable salt of a compound as described herein can alternatively be formed during the synthesis of the compound, e.g., in the course of isolating the compound from a reaction mixture or re-crystallizing the compound.
  • a pharmaceutically acceptable salt of the compounds described herein may optionally be an acid addition salt and/or a base addition salt.
  • An acid addition salt comprises at least one basic (e.g., amine and/or guanidinyl) group of the compound which is in a positively charged form (e.g., wherein the basic group is protonated), in combination with at least one counter-ion, derived from the selected acid, that forms a pharmaceutically acceptable salt.
  • the acid addition salts of the compounds described herein may therefore be complexes formed between one or more basic groups of the compound and one or more equivalents of an acid.
  • a base addition salt comprises at least one acidic (e.g., carboxylic acid) group of the compound which is in a negatively charged form (e.g., wherein the acidic group is deprotonated), in combination with at least one counter-ion, derived from the selected base, that forms a pharmaceutically acceptable salt.
  • the base addition salts of the compounds described herein may therefore be complexes formed between one or more acidic groups of the compound and one or more equivalents of a base.
  • the acid additions salts and/or base addition salts can be either mono-addition salts or poly-addition salts.
  • addition salt refers to a salt in which the stoichiometric ratio between the counter-ion and charged form of the compound is 1:1, such that the addition salt includes one molar equivalent of the counter-ion per one molar equivalent of the compound.
  • poly- addition salt refers to a salt in which the stoichiometric ratio between the counter-ion and the charged form of the compound is greater than 1:1 and is, for example, 2: 1, 3: 1, 4: 1 and so on, such that the addition salt includes two or more molar equivalents of the counter-ion per one molar equivalent of the compound.
  • An example, without limitation, of a pharmaceutically acceptable salt would be an ammonium cation or guanidinium cation and an acid addition salt thereof, and/or a carboxylate anion and a base addition salt thereof.
  • the base addition salts may include a cation counter-ion such as sodium, potassium, ammonium, calcium, magnesium and the like, that forms a pharmaceutically acceptable salt.
  • the acid addition salts may include a variety of organic and inorganic acids, such as, but not limited to, hydrochloric acid which affords a hydrochloric acid addition salt, hydrobromic acid which affords a hydrobromic acid addition salt, acetic acid which affords an acetic acid addition salt, ascorbic acid which affords an ascorbic acid addition salt, benzenesulfonic acid which affords a besylate addition salt, camphorsulfonic acid which affords a camphorsulfonic acid addition salt, citric acid which affords a citric acid addition salt, maleic acid which affords a maleic acid addition salt, malic acid which affords a malic acid addition salt, methanesulfonic acid which affords a methanesulfonic acid (mesylate) addition salt, naphthalenesulfonic acid which affords a naphthalenesulfonic acid addition salt, oxalic acid which affords an oxalic acid addition salt,
  • prodrug refers to a compound which is converted in the body to an active compound (e.g., the compound of the formula described hereinabove).
  • a prodrug is typically designed to facilitate administration, e.g., by enhancing absorption.
  • a prodrug may comprise, for example, the active compound modified with ester groups, for example, wherein any one or more of the hydroxyl groups of a compound is modified by an acyl group, optionally (Ci-4)-acyl (e.g., acetyl) group to form an ester group, and/or any one or more of the carboxylic acid groups of the compound is modified by an alkoxy or aryloxy group, optionally (Ci-4)-alkoxy (e.g., methyl, ethyl) group to form an ester group.
  • an acyl group optionally (Ci-4)-acyl (e.g., acetyl) group to form an ester group
  • any one or more of the carboxylic acid groups of the compound is modified by an alkoxy or aryloxy group, optionally (Ci-4)-alkoxy (e.g., methyl, ethyl) group to form an ester group.
  • each of the compounds described herein, including the salts thereof, can be in a form of a solvate or a hydrate thereof.
  • solvate refers to a complex of variable stoichiometry (e.g., di-, tri-, tetra-, penta-, hexa-, and so on), which is formed by a solute (the heterocyclic compounds described herein) and a solvent, whereby the solvent does not interfere with the biological activity of the solute.
  • solute the heterocyclic compounds described herein
  • hydrate refers to a solvate, as defined hereinabove, where the solvent is water.
  • the compounds described herein can be used as polymorphs and the present embodiments further encompass any isomorph of the compounds and any combination thereof.
  • the compounds and structures described herein encompass any stereoisomer, including enantiomers and diastereomers, of the compounds described herein, unless a particular stereoisomer is specifically indicated.
  • enantiomer refers to a stereoisomer of a compound that is superposable with respect to its counterpart only by a complete inversion/reflection (mirror image) of each other. Enantiomers are said to have “handedness” since they refer to each other like the right and left hand. Enantiomers have identical chemical and physical properties except when present in an environment which by itself has handedness, such as all living systems.
  • a compound may exhibit one or more chiral centers, each of which exhibiting an (R) or an (S) configuration and any combination, and compounds according to some embodiments of the present invention, can have any their chiral centers exhibit an (R) or an (S) configuration.
  • diastereomers refers to stereoisomers that are not enantiomers to one another. Diastereomerism occurs when two or more stereoisomers of a compound have different configurations at one or more, but not all of the equivalent (related) stereocenters and are not mirror images of each other. When two diastereoisomers differ from each other at only one stereocenter they are epimers. Each stereo-center (chiral center) gives rise to two different configurations and thus to two different stereoisomers.
  • embodiments of the present invention encompass compounds with multiple chiral centers that occur in any combination of stereo-configuration, namely any diastereomer.
  • compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range.
  • a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range.
  • the phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.
  • method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • treating includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
  • HTS High-throughput screening
  • the screen was performed in sterile tissue culture-treated white 384 flat bottom well plates (Greiner, #781080). 8xl0 3 HeLa cells were seeded in each well and incubated at 37 °C in 5% CO 2 humidified incubator for 4 h to let the cells adhere. Overall, 39,118 small molecules that were obtained from diverse bioactive compound libraries were added to the wells at a final concentration of 10 ⁇ M using the Multidrop Combi Reagent Dispenser (Thermo-Fisher Scientific).
  • the ability of the top 8 hits to inhibit Salmonella Typhimurium (STM) invasion into HeLa cells was determined by the bioluminescence assay as described above ( Figure 3 left panel). Salmonella infection in the presence of the 8 compounds at a final concentration of 10 ⁇ M is shown relative to its infection in the absence of the compounds. Cytochalasin D (CD) that blocks actin polymerization and Salmonella invasion was used as a positive control. ( Figure 3 right panel) the effect of the compounds on Salmonella infection was tested independently by the gentamycin protection assay in the presence and absence of the compounds. Host cells infection by S. Typhimurium (STM) in the absence of compounds was used as a reference. STM isogenic mutants invA and ssaR that are impaired in their ability to invade and replicate in host cells, respectively were used as genetic negative controls.
  • STM Salmonella Typhimurium
  • FIG. 3 shows using two independent approaches that all eight top hits can effectively inhibit epithelial cells infection by S. Typhimurium.
  • EXAMPLE 4 shows using two independent approaches that all eight top hits can effectively inhibit epithelial cells infection by S. Typhimurium.
  • Figure 4 shows that all eight compounds are not cytotoxic to HeLa nor to HB2 cells at 10 ⁇ M concentration.
  • Typhimurium culture expressing ssek3::lux was subcultured 1 : 100 in LB medium for 3 h at 37 °C on a shaker incubator to the late logarithmic phase (ODeoo ⁇ 1).
  • Salmonella culture was then diluted 1:50 in DMEM and aliquots of 100 pl were added to each well to reach an MOI of 1:30 (cells per bacteria) and incubated at 37 °C 5% CO 2 to allow infection.
  • One h post-infection, gentamicin was added to a final concertation of 20 pg/ml and the plates were incubated for overnight.
  • Figure 5 shows the inhibitory activity of the eight compounds in a dose-dependent manner and its ability to reduce host cell infection by S. Typhimurium.
  • the identified compounds inhibit Salmonella intracellular replication rather than invasion Reducing pathogen infection can be mediated either by inhibiting its invasion into host cells or by limiting its intracellular survival/ replication.
  • 5xl0 4 HeLa cells were seeded in 24- well (Greiner, #662160), cell culture-treated, flat-bottom microplate and incubated at 37° C in a humidified 5% CO 2 incubator for 24 h prior to infection.
  • the medium was replaced with fresh DMEM medium containing the PCM-0094889, PCM- 0086166, PCM-0004846, PCM-0103431, PCM-0095494, PCM-0095293, PCM-0001349, and PCM-0095564 compounds at a final concentration of 10 ⁇ M, which were incubated with the cells for 3 h.
  • An overnight S. Typhimurium SL1344 culture was subcultured 1 : 100 into fresh LB medium and grown at 37 °C on a shaker incubator to the late logarithmic phase. After 3 h, the bacteria were diluted 1 : 100 in DMEM and 500 pl from the diluted bacteria were added into each well at MOI ⁇ 1:50.
  • the infected cells were spun down for 5 min at 1000 g and then incubated at 37 °C 5% CO 2 . After 10 min, cells were washed three times with PBS and fresh medium supplemented with 10 ⁇ M of each compound were added to each well and further incubated for 20 min. At 30 min post infection, the medium was replaced with a fresh medium containing 100 pg/ml gentamicin and 10 ⁇ M of each compound and incubated for 90 min. At 2 h post infection, cells were washed three times with PBS and lysed with 250 pl lysis buffer (0.1% SDS, 1% Triton X-100 in PBS) by incubating the cells for 10 min at room temperature with gentle agitation.
  • Intracellular fold replication was determined by the ratio between the number of intracellular cells counted at 24 h post infection and their number at 2 h post infection.
  • S. Typhimurium replication in the presence of the inhibitors was calculated relative to its replication in the absence of the compounds.
  • S. Typhimurium ssaR null mutant, which is impaired in host cells replication was included as a positive control.
  • the charts show the mean and standard error of the mean (SEM) of seven biological repeats in two independent experiments.
  • Figure 6 shows that all eight compounds can inhibit Salmonella intracellular replication rather than invasion and that this activity of the compounds limits the ability of the pathogen to infect and thrive within host cells.
  • BMDMs bone marrow derived macrophages
  • Bone marrow-derived macrophage(BMDMs) were isolated from the femur leg bone of 7- week old SWISS female mice. Macrophages were diluted in BMDM medium [50% DMEM high glucose, 20% FBS, 30% L-929 conditioned medium, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 nM ⁇ -mercaptoethanol] and seeded at 2.5xl0 5 cells/ml in a 24-well, cell culture-treated dish, 24 h prior to bacterial infection.
  • BMDM medium 50% DMEM high glucose, 20% FBS, 30% L-929 conditioned medium, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 nM ⁇ -mercaptoethanol
  • BMDM medium was replaced by BMDM fresh medium supplemented with 10 ⁇ M of the compounds (PCM-0094889, PCM-0086166, PCM- 0004846, PCM-0103431, PCM-0095494, PCM-0095293, PCM-0001349, and PCM-0095564) and incubated for 3 h at 37 °C under 5% CO 2 atmosphere.
  • Macrophages were infected at an MOI of 1:10 with cultures of S. Typhimurium SL1344 and its ⁇ ssR isogenic mutant that is impaired in intracellular replication as a negative control. Infected cells were spun down at 1000 g for 5 min and incubated for 30 min at 37 °C, 5% CO 2 .
  • Infection experiments were carried out using the gentamicin protection assay. At 30 min p.i, cells were washed three times with PBS to remove extracellular bacteria, and medium containing 100 pg/ml gentamicin was added for 1 h incubation. Wells were then washed three times with PBS and medium was replaced with fresh BMDM containing 10 pg/ml gentamicin. To determine intracellular growth of Salmonella at 2 and 20 h p.i., cells were washed three times with PBS and lysed with 250 pl of lysis buffer (0.1% SDS, 1% Triton X- 100 in PBS).
  • the number of CFUs in each well was quantified by plating serial dilutions of cell lysates on selective LB-agar plats.
  • Salmonella uptake (left panel) was calculated as the number of intracellular bacteria recovered at 2 h p.i., divided by the infecting inoculum (CFUs).
  • Salmonella survival (right panel) was calculated as the number of intracellular bacteria recovered at 24 h p.i., divided by intracellular bacteria recovered at 2 h p.i.
  • the charts show the mean and standard error of the mean (SEM) of three biological repeats.
  • Figure 7 shows that compounds 1-4 can effectively inhibit S. Typhimurium replication in mouse BMDM by more than 50 %.
  • the identified compounds can inhibit the intracellular growth of Listeria monocytogenes
  • Cell infection at am MOI of 1:50 was conducted by adding 20 pl of Listeria suspension to each well, following by incubation at 37 °C under 5% CO 2 atmosphere. 30 min p.i. the cells were washed with PBS and the medium was replaced with fresh medium supplemented with 10 ⁇ M of the tested compounds. One h p.i., gentamycin was added to each well at a final concentration of 50 ⁇ g/ml. At 6 h p.i., the cells were washed with PBS and lysed with 250 pl DDW at room temperature. The cell lysates were then diluted in PBS and plated on LB plates for CFU count.
  • Intracellular Listeria infection was calculated as the number of intracellular bacteria recovered at 6 h p.i., divided by the infecting inoculum (CFUs). Listeria infection in the presence of the compounds is shown relative to the number of intracellular bacteria that were recovered from cells that were not treated with the compounds.
  • the charts show the mean and standard error of the mean (SEM) of six biological repeats in two independent experiments, biological repeats.
  • Figure 8 shows that compounds 1, 4, 5, 7, and 8 were able to actively inhibit Listeria monocytogenes infection of HeLa cells.
  • L. monocytogenes and S. Typhimurium are Gram-positive and Gram-negative bacteria, suggests that these compounds are active against very different bacteria and have the potential to inhibit infection by a wide range of pathogens, most likely via a host-dependent mechanism.
  • PCD programmed cell death
  • 4.6xl0 4 HeLa cells were seeded in each well of a 96-well, cell culture-treated, flat-bottom white microplate (Greiner, #665083) and incubated at 37 °C 5 % CO 2 humidified incubator for 4 h to let the cells adhere, in the presence of 10 ⁇ M of compounds PCM-0103431 and PCM-0001349 and their chemical derivatives. As a positive control 2 ⁇ M Cytochalasin D (CD) was also included. Cells were infected with an overnight cultures of S.
  • CD Cytochalasin D
  • Typhimurium culture expressing the reporter system ssek3::lux which was subcultured 1:100 in LB medium for 3 h at 37 °C and grown to the late logarithmic phase (OD 600 ⁇ 1).
  • Salmonella culture was then diluted 1 :50 in DMEM and aliquots of 100 pl were added to each well to reach an MOI of 1:50 (cells per bacteria) and incubated at 37 °C, 5% CO 2 to allow infection.
  • One h p.i, gentamicin was added to a final concertation of 20 pg/ml and the infected cells were incubated for overnight.
  • FIG 10A shows that chemical derivatives of compounds 4 and 7 can improve the inhibitory function of these compounds. Intracellular infection is shown relative to the Salmonella infection of HeLa cells in the presence of 0.1% DMSO. The charts show the mean and SEM of three biological repeats in three independent experiment. Chemicals derivatives of compounds PCM-0103431 and PCM- 0001349 that presented enhanced inhibitory activity are highlighted by a red box, and their chemical structures are shown in Figure 10B.
  • R 65 istant Strains of Mycobacterium tuberculosis by Inhibition of Src Family Kinases Lowers Disease Burden and Pathology. mSphere 2016; 1. J ON. Review on Antimicrobial R 65 istance. Antimicrobial R 65 istance: Tackling a Crisis for the Health and Wealth of Nations. 2014. Kamaruzzaman NF, Kendall S, Good L. Targeting the hard to reach: challenges and novel strategies in the treatment of intracellular bacterial infections. Br J Pharmacol 2017; 174:2225- 36. Li L, Dickinson MS, Coers J, Miao EA. Pyroptosis in defense against intracellular bacteria. Semin Immunol 2023; 69:101805. Petit TJP, Lebreton A.
  • R 65 istant: A Major Challenge in Clinical Diagnosis. Infect Disord Drug Targets 2023. Kellermann M, Scharte F, Hensel M. Manipulation of Host Cell Organelles by Intracellular Pathogens. Int J Mol Sci 2021; 22. Liu Y, Jia Y, Yang K, Wang Z. Heterogeneous Strategies to Eliminate Intracellular Bacterial Pathogens. Front Microbiol 2020; 11:563.

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Abstract

L'invention concerne des compositions et des méthodes de traitement ou de prévention d'une infection par un pathogène intracellulaire chez un sujet en ayant besoin.
PCT/IL2024/050636 2023-06-28 2024-06-28 Traitement d'infections provoquées par des pathogènes intracellulaires Pending WO2025004049A1 (fr)

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